Journal of Microencapsulation, October 2008; 25(7): 499–512

Vesicular aceclofenac systems: A comparative study between

liposomes and niosomes

MAHA NASR1, SAMAR MANSOUR1, NAHED D. MORTADA1,

& A. A. ELSHAMY2
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1
Department of Pharmaceutics and 2Department of Drug Technology, Faculty of Pharmacy,
Ain Shams University, Cairo, Egypt

(Received 15 July 2007; accepted 13 March 2008)

Abstract
Vesicular delivery systems have been reported to serve as local depot for sustained drug release. Aceclofenac multilamellar
liposomes and niosomes were prepared and a comparative study was done between them through evaluation of entrapment
efficiency, particle size, shape, differential scanning calorimetry and in vitro drug release. A stability study was carried out
by investigating the leakage of aceclofenac and the change in the vesicles particle size when stored at (2–8 C) for 3 months.
For personal use only.

The anti-inflammatory effect of aceclofenac vesicles was assessed by the rat paw oedema technique. Results showed that the
entrapment efficiency and the in vitro release of aceclofenac from the vesicles can be manipulated by varying the cholesterol
content, the type of surfactant as well as the type of charge. Niosomes showed better stability than liposomes. Both vesicular
systems showed significant sustained anti-inflammatory activity compared to the marketed product, with niosomes being
superior to liposomes as manifested by both oedema rate and inhibition rate percentages suggesting their effectiveness as topical
anti-inflammatory delivery systems.

Keywords: Aceclofenac, liposomes, niosomes, rat paw oedema, physical stability

Introduction valuable biocompatible drug delivery system (Pavelic

Aceclofenac is a phenylacetic acid derivative related to
diclofenac. It is a practically insoluble in water drug
with a molecular weight of 354.19, a pKa value of 4.7
and a log p-value of 1.23 (Alvarez-Larena et al. 1992;
British Pharmacopoeia 1998). It is a potent analgesic,
anti-pyretic and anti-inflammatory agent used in the
management of moderate-to-severe pain and in
rheumatic disorders such as rheumatoid arthritis and
ankylosing spondylitis (Pasero et al. 1995; Martel-
Pelletier et al. 1997). When taken orally, aceclofenac
induce the same side effects of other NSAIDs including
those related to GIT, liver and kidney function as well
as disturbance of platelets function (Insel 1992).
Vesicular delivery systems have proved superiority
over conventional dosage forms, especially for the
topical route of administration (Egbaria and Weiner
1991). Liposomal formulations have been widely
administered for topical treatment of diseases as a
et al. 2001). Local anaesthetic agents, anti-
inflammatory agents, anti-fungal agents, antibiotics,
vitamins and peptide or proteins are among the
substances whose liposomal application holds promise
when applied topically for localized drug delivery
(Rolland 1993). However, despite that the majority of
reports have concerned the use of phospholipid vesicles
or liposomes; they exhibit certain disadvantages, one of
which relates to their chemical instability. The cost and
variable purity of natural phospholipids might lead
to other considerations. Therefore, alternatives to
phospholipids are the focus of interest from a technical
view point (Baillie et al. 1985).
Non-ionic surfactant vesicles (niosomes) have been
widely studied as an alternative to liposomes. They are
biodegradable, biocompatible, non-toxic and capable of
encapsulating large quantities of material in a relatively
small volume of vesicles. In fact they offer several
advantages over liposomes: higher chemical stability,

Correspondence: Maha Nasr, MSc, Faculty of Pharmacy, Department of Pharmaceutics, Ain Shams University, Cairo, Egypt. E-mail: maha2929@gmail.com

ISSN 0265–2048 print/ISSN 1464–5246 online ß 2008 Informa UK Ltd.

DOI: 10.1080/02652040802055411
 500 M. Nasr et al.

Table I. Entrapment efficiency, particle size and T8h of multilamellar liposomes.

Liposomal formulations
(PC:CH) or (PC:CH:DP or SA) Entrapment efficiency* Distribution modal Polydispersity T8h2
(molar ratio) (%  SD) size (mm) (SPAN index)1 (%  SD)

PC:CH (7:4) 29.9  1 0.88 0.34 61.9  0.2

PC:CH (7:6) 31.8  0.6 0.70 0.43 53.5  1.4
PC:CH (7:7) 19.5  0.2 0.59 0.14 43.4  0.7
PC:CH:SA (7:4:1) 45.5  0.4 1.33 0.6 50.7  2.6
PC:CH:SA (7:6:1) 47.2  0.2 1.10 0.19 38.2  0.6
PC:CH:SA (7:7:1) 32.4  0.1 1.09 0.53 35.8  2
PC:CH:DP (7:4:1) 37.9  0.2 1.35 0.26 84.4  1.4
PC:CH:DP (7:6:1) 39.6  1 1.09 0.4 64.8  1.3
PC:CH:DP (7:7:1) 30.9  1.5 1.08 0.45 58.7  3

Notes: PC: phosphatidylcholine; CH: cholesterol; SA: stearylamine, DP: dicetylphosphate.

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*Each value is an average of three determinations.

1
Polydispersity/SPAN ¼ (particle diameter at 90% cumulative size – particle diameter at 10% cumulative size)/(particle diameter at 50%
cumulative size).
2
T8h: cumulative percentage released of aceclofenac after 8 h.

intrinsic skin penetration enhancing properties and Preparation of aceclofenac multilamellar liposomes
lower costs (Baillie et al. 1985; Uchegbu and
Aceclofenac multilamellar liposomal formulations were
Florence 1995; Uchegbu and Vyas 1998; Vora et al.
prepared using the thin film hydration technique
1998). Niosomes behave in vivo like liposomes, prolong-
(Gabriels and Plaizier-Vercammen 2003; Vyas et al.
ing the circulation of entrapped drug and altering its
2004). The composition of the formulations is shown
organ distribution and metabolic stability (Ruckmani
in Table I. The drug (50 mg) and the lipid components
For personal use only.

et al. 2000).
The aim of this work was to formulate aceclofenac
multilamellar liposomal and niosomal formulations
using a thin film hydration method intended for topical
administration on the skin. The prepared formulations
were characterized and evaluated for their in vitro
release performance. The anti-inflammatory activity of
selected formulations was evaluated and compared to
that of the marketed product.
Materials and methods
Materials
Acelofenac was kindly provided by Bristol-Myers
Squibb Company (Cairo, Egypt). L-- Phosphatidyl
choline, type X-E: from dried egg yolk (PC), cholesterol
(CH), stearylamine (SA), dicetyl phosphate (DP) and
carrageenan were purchased from Sigma Chemical Co.
(St. Louis, USA). Sorbitan monopalmitate (Span 40)
and sorbitan monostearate (Span 60) were purchased
from Fluka Chemical Co. (Buchs, Switzerland).
Methanol, chloroform, absolute ethyl alcohol,
potassium dihydrogen phosphate, disodium hydrogen
phosphate and sodium chloride were purchased from
Adwic, El-Nasr chemical Co. (Cairo, Egypt) according
to the methods of Prolabo (Paris, France). Acetyl uranil
(Uranyl acetate-2-hydrate) was purchased from Allied
Signal (Riedel-dahaen, Germany). Spectra/Por dialysis
membrane, 12.000–14.000 molecular weight cut-off,
was purchased from Spectrum Laboratories Inc.
(Rancho Dominguez, Canada).
(200 mg), including the egg phosphatidylcholine (PC)
and cholesterol (CH), either alone or mixed with charge
inducing agents: stearylamine (SA) or dicetyl phosphate
(DP), in different molar ratios were accurately
weighed into a 250 ml long-neck quick fit round
bottom flask and dissolved in chloroform:methanol
mixture (2:1, v/v). The organic solvent system was
slowly removed under reduced pressure, using a rotary
evaporator (Janke and Kunkel, model RVO5-ST, IKA
Laboratories, Staufen, Germany) at 40 C and 150 rpm
(Agarwal el al. 2001; Bhatia et al. 2004) such that a thin
film of dry lipids was formed on the inner surface of the
flask. The dry lipid film was slowly hydrated with 10 ml
of phosphate buffered saline (PBS) pH 7.4 for 1 h.
The resulting liposomal suspension was mechanically
shaken for 1 h using a thermostatically controlled
mechanical shaker (Kottermann, Hanigsen, Germany)
at 60 rpm and 40 C leading to the formation of
multilamellar liposomes. The liposomal suspension
was left to mature overnight at 4 C, to ensure hydration
of the lipid.
Preparation of aceclofenac multilamellar niosomes
Aceclofenac multilamellar niosomes were also prepared
using the thin film hydration method. The composition
of the formulations is shown in Table II. Accurately
weighed quantities of the drug (50 mg), the surfactant
(either Span 40 or Span 60) and CH in different molar
ratios were dissolved in chloroform/methanol mixture
(2 : 1, v/v) in a round-bottom flask following the same
procedure previously described for liposomes with the
difference that the organic solvents were removed

Table II. Entrapment efficiency, particle size and T8h of multilamellar niosomes.
Niosomal formulation Entrapment efficiency*
composition (molar ratio) (%  SD)
Span 40:CH (7:4) 27.7  0.6
Span 40:CH (7:6) 32.5  0.1
Span 40:CH (7:7) 14.2  0.2
Span 60:CH (7:4) 31.6  0.2
Span 60:CH (7:6) 45.1  0.5
Span 60:CH (7:7) 37.9  0.4
Notes: CH: cholesterol.
*Each value is an average of three determinations.
1
Polydispersity/SPAN ¼ (particle diameter at 90% cumulative size – particle diameter at 10% cumulative size)/(particle diameter at 50%
cumulative size).
2
T8h: cumulative percentage released of aceclofenac after 8 h.
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under vacuum in the rotary evaporator at 60 C
(Hao et al. 2002; Manconi et al. 2002).
Determination of aceclofenac entrapment efficiency
Aceclofenac liposomes and niosomes were separated
from free unentrapped drug by centrifugation at
13 000 rpm for 30 min. The pellets formed were
washed twice each with 10 ml phosphate buffered
saline and recentrifuged again for 30 min (Morilla
For personal use only.
et al. 2002). The prepared liposomal and niosomal
formulations were decanted and kept in the refrigerator
(Skalko-Basnet et al. 2000). The concentration of the
entrapped drug was determined by lysis of an aliquot of
100 ml of the liposomal or niosomal formulations with
absolute alcohol and sonication to obtain a clear
solution (Law et al. 1994; Fang et al. 2001a). The
concentration of aceclofenac in absolute alcohol was
determined spectrophotometrically at 276 nm using UV
spectrophotometer (Shimadzu, model UV-1601 PC,
Kyoto, Japan). No interference from liposomal or
niosomal components was found at this wavelength.
Further dilution was made if necessary. The encapsula-
tion or entrapment efficiency was calculated through
the following relationship (Aggarwal and Kaur 2005):
Entrapment efficiency percentage
Entrapped drug
¼  100
Total drug
Characterization of aceclofenac liposomes and niosomes
Photomicroscopic analysis and transmission electron
microscopy (TEM). Samples of the positively charged
aceclofenac liposomes composed of PC:CH:SA (7:4:1)
molar ratio and aceclofenac niosomes composed of
Span 60:CH (7:4) molar ratio were used.
For microscopic investigation, the vesicles were
examined under a binocular optical microscope
(Carl Zeiss, Berlin, Germany) and photographed at a
magnification of 400 (Gabriels and Plaizier-
Vercammen 2003) by means of a fitted camera
(Panasonic, Japan) for morphological evaluation.
Vesicularal aceclofenac systems 501
Distribution modal Polydispersity T8h2
size (mm) (SPAN index)1 (%  SD)
0.70 0.38 58.2  3.2
0.84 0.44 52.1  0.4
0.84 0.43 46.1  1.4
0.85 0.66 49.4  2.7
0.89 0.14 43.3  2.2
1.04 0.51 36.2  0.4
Transmission electron microscopy (Model
JEM–100S, Joel, Tokyo, Japan) was also used to
examine the vesicle ultrastructure (Maestrelli et al.
2005). Negatively stained samples were prepared by
placing a drop of 1 ml of the vesicular suspension on a
carbon coated grid (Ahn et al. 1995). The suspension
was left for 2 min, to allow its absorption in the carbon
film, and the excess liquid was drawn off with filter
paper. Subsequently, a drop of 2% (w/v) acetyl uranil
aqueous solution was placed on the grid. The excess
was removed with distilled water and the samples were
examined by TEM at 20 and 25 kV (Taylor et al. 1990;
Nagarsenker et al. 1999; Manconi et al. 2003).
Determination of vesicle size. The size of the prepared
aceclofenac vesicles was determined by light
scattering based on laser diffraction using the
Malvern Mastersizer (Malvern Instruments Ltd.,
Worcestershire, UK) (Joshi and Misra 2001). The
polydispersity, i.e. the width of the particle size
distribution was given by a SPAN index which was
calculated by the following equation (Tabbakhian
et al. 2006):
Polydispersity ðSPANÞ ¼ ðD0:9  D0:1 Þ=D0:5 ð2Þ
where D0.9, D0.1 and D0.5 are the particle diameters
ð1Þ
determined at the 90th, 10th and 50th percentile of
particles undersized, respectively.
Differential scanning calorimetry (DSC). DSC experi-
ments were performed with a differential scanning
calorimeter (Schimadzu, model TA-50 WSI, Kyoto,
Japan) calibrated with indium. Samples of aceclofenac,
plain and drug loaded multilamellar liposomes
composed of PC:CH:SA (7:6:1) molar ratio as well as
multilamellar niosomes of Span 60:CH (7:6) molar ratio
were submitted to DSC analysis. The analyses were
carried out on 40 ml or 1 mg samples sealed in
standard aluminum pans. Thermograms were
obtained at a scanning rate of 10 C min1 using dry
 502 M. Nasr et al.
nitrogen flow (25 ml min1). Isotonic phosphate buffer
(pH 7.4) was employed as a reference (Ganesan et al.
1984; Fresta et al. 1998).
In vitro release of aceclofenac from liposomes and
niosomes. The release of aceclofenac from liposomal
and niosomal formulations was determined using the
membrane diffusion technique (Devaraj et al. 2002;
Glavas-Dodov et al. 2002). An accurately measured
amount of aceclofenac vesicular formulations, equiva-
lent to 2 mg aceclofenac, was suspended in 1 ml PBS
pH 7.4. The suspension was transferred to a glass
cylinder having a length of 7 cm and diameter of 2.5 cm
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previously covered at its lower end with a soaked
cellulose membrane (Spectra/Por dialysis membrane
12–14 000 Mw cut-off). The cylinder was placed in the
dissolution flask of a USP dissolution tester (Pharma
Test, Hainburg, Germany) containing 100 ml PBS pH
7.4. The cylinder was allowed to rotate at a constant
speed (50 rpm). The phosphate buffered saline was
maintained at a temperature of 32 C. At pre-deter-
mined time intervals (0.25, 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h),
samples were withdrawn and assayed spectrophotome-
trically at 274 nm for the drug content. Each withdrawn
sample was replaced by an equal volume of PBS
For personal use only.
(Ruckmani et al. 2000). The results were the mean of
three runs. The obtained data were kinetically treated to
determine the order of release.
Physical stability. The selected liposomal and nioso-
mal batches were sealed in 20 ml glass vials and stored
as pellets (2–8 C) (Patel and Misra 1999). Samples from
each batch were withdrawn at specified time intervals to
determine the residual amount of the drug in the
vesicles as described previously (Shahiwala and Misra
2002; Sinico et al. 2005). The leaked out drug during the
storage period was separated by dilution of an aliquot
of each formulation with phosphate buffered saline and
centrifugation at 13 000 rpm for 30 min. At the end of
the storage period, particle size was also determined at
the end of the storage period; results of which can be
taken as an index of stability (Yan-yu et al. 2006).
In vivo study of liposomes and niosomes. This work will
include the estimation of the anti-inflammatory activity
of the selected aceclofenac liposomes and niosomes
compared to plain vesicles as well as to the marketed
product using the rat paw oedema test. The protocol of
the present work was approved by Experiments
and Advanced Pharmaceutical Research Unit
(EAPRU), Faculty of Pharmacy, Ain Shams
University, Cairo, Egypt.
Adult male albino rats, weighing 130–150 g, were
divided into eight groups of five animals each, namely:
Group I received topical saline application, Group II
received plain liposomes, Group III received plain
niosomes, Group IV received aceclofenac liposomes
composed of PC:CH:SA (7:4:1) molar ratio,
respectively, Group V received aceclofenac liposomes
composed of PC:CH:SA (7:6:1) molar ratio, respec-
tively, Group VI received aceclofenac niosomes com-
posed of Span 60:CH (7:4) molar ratio, respectively,
Group VII received aceclofenac niosomes composed of
Span 60:CH (7:6) molar ratio, respectively, and Group
VIII received the marketed Bristaflam creamÕ . The
volume of paw oedema (ml) was measured in each
animal using a plethysmometer (UGO-Basile, 7140,
Comerio, Italy) to a precision of two decimal places.
The rats were marked on the left hind paw just beyond
the tibiotarsal junction, so that every time the paw was
dipped in the electrolyte fluid column up to a fixed
mark to ensure constant paw volume.
The tested formulations were applied to the left hind
paws of rats using an amount from each of them
equivalent to 1 mg of aceclofenac. The area of applica-
tion was occluded with a parafilm (Nagarsenker and
Joshi 1997) and was left in place for 2 h. The parafilm
was then removed and the residual formula on the
surface was wiped off (Shahiwala and Misra 2002).
After 2 h of topical application, initial paw volume of
the rats was measured by dipping the rat paw into the
electrolyte column. A volume of 0.1 ml of 1% (w/v)
carrageenan solution in saline was then injected in the
sub-plantar region of left hind paw of the rats and the
increase in volume due to fluid displacement was noted
from a digital display (Penzes et al. 2005). Measurement
of paw volume was done after 1, 2, 3, 4, 5, 6, 7 and 8 h.
The oedema and inhibition rate percentage of each
group was calculated as follows:
Vt  V0
Oedema rate ðE%Þ ¼  100 ð3Þ
V0
Ec  Et
Inhibition rate ðI%Þ ¼  100 ð4Þ
Ec
where V0 is the mean paw volume before carrageenan
injection (ml), Vt is the mean paw volume after
carrageenan injection (ml), Ec is the oedema rate of
control group and Et is the oedema rate of the treated
group (Mei et al. 2003, 2005).
Data were expressed as mean  SD and statistically
assessed by one way analysis of variance (ANOVA).
Values for oedema rate percentage for liposomal
formulations, niosomal formulations and the marketed
product were compared to the saline control and the
differences were determined statistically using
Dunnett’s t-test.
Results and discussion
Entrapment efficiency for liposomal formulations
Effect of cholesterol content. Egg phospholipids were
chosen rather than soya phospholipids for the
preparation of liposomes because previous results

have shown that they exhibited significantly higher
hydration effects than soya phospholipids; therefore,
they are recommended for the preparation of topical
formulations (Betz et al. 2005).
Data in Table I reveals that increasing the CH
content in liposomal formulations significantly
increased the entrapment efficiency of aceclofenac up
to an optimum PC:CH ratio (7:6). The percentage
entrapment efficiency significantly increased from
29.9% to 31.8% for neutral liposomes composed of
7:4 and 7:6 PC:CH molar ratios, respectively, but
it significantly decreased to 19.5% with liposomal
formulations of 7:7 PC:CH molar ratio (p 5 0.05).
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The increase in entrapment efficiency is explained on
the basis of recognized effects for cholesterol: increasing
the microviscosity and rigidity of the lipidic bilayer,
resulting in a higher stability (Moribe et al. 1999),
reduced permeability (Du Plessis et al. 1996) of the
liposomal membrane and hence greater drug retention
(Joshi and Misra 2001). However, further increase in
CH content beyond a certain level (7:6 PC:CH molar
ratio) starts disrupting the regular bilayered structure
leading to lowering of drug entrapment levels (Patel and
Misra 1999).
For personal use only.
Charge inducing agents. Stearylamine and dicetyl
phosphate were used as positive and negative charge
inducers, respectively. Data in Table I reveals that the
charged liposomes exhibited an increased entrapment
of aceclofenac compared to neutral ones (Sharma et al.
1994). The percentages encapsulation efficiencies
significantly increased from 29.9% for neutral
liposomes composed of PC:CH (7:4) molar ratio to
45.5% and 37.9% for their positively and negatively
charged counterparts, respectively, from 31.8% for
neutral liposomes of PC:CH (7:6) molar ratio to 47.2%
and 39.6% for their positively and negatively charged
counterparts, respectively, and from 19.5% for neutral
liposomes composed of PC:CH (7:7) molar ratio to
32.4% and 30.9% for their positively and negatively
charged counterparts, respectively (p 5 0.05).
The incorporation of charged lipids into bilayers
increases the volume of the aqueous compartments by
separating adjacent bilayers due to charge repulsion,
resulting in an increase in the interlamellar resistance
between successive bilayers and an increase in trapped
volume, hence increase in encapsulation (Kulkarni
et al. 1995; DuPlessis et al. 1996). Moreover, data in
Table I reveals that positively charged liposomes
exhibited the highest entrapment efficiency followed
by negatively charged ones then neutral ones, using the
same PC:CH molar ratio. Aceclofenac is a weak acid
and an electrostatic attraction would occur between
drug anion and the positively charged SA, this
attraction would account for the highest entrapment
efficiency obtained with aceclofenac positively charged
liposomal formulations (El-Gazayerly and Hikal 1997).
Vesicularal aceclofenac systems 503
Entrapment efficiency for niosomal formulations
Cholesterol content. Data in Table II shows that the
entrapment efficiency percentages of aceclofenac
in niosomal formulations were 27.7%, 32.5% and
14.2% for multilamellar niosomes composed of 7:4,
7:6 and 7:7 Span 40:CH molar ratios, respectively.
On the other hand, the percentages of entrapment
efficiency of aceclofenac were 31.6%, 45.1%, 37.9% for
multilamellar niosomes composed of 7:4, 7:6 and 7:7
Span 60:CH molar ratios, respectively. As observed
with liposomal formulations, the incorporation of
CH into niosomes significantly increased the drug
entrapment efficiency up to an optimum Span:CH
ratio (7:6) (p 5 0.001). Cholesterol alters the fluidity of
chains in bilayers and, when present in sufficient
concentration, abolishes the gel to liquid phase
transition of surfactant bilayers (Uchegbu and
Florence 1995; Devaraj et al. 2002, Hao et al. 2002).
It also increases the microviscosity of niosomal
membrane conferring more rigidity (Manosroi et al.
2003). Further increase of CH content to reach 7:7
Span:CH molar ratio significantly reduced the entrap-
ment efficiency. This could be interpreted similarly as in
the case of aceclofenac liposomes based on the effect of
cholesterol on the rigidity of the regular bilayered
structure leading to loss of drug entrapment levels
(Agarwal el al. 2001; Guinedi et al. 2005).
The type of surfactant. An increasing number of
non-ionic surfactants have been found to formulate
niosomes among which sorbitan esters are much more
common. It was reported that the length of alkyl chain
is a crucial factor of permeability and that long chain
surfactants produce high drug entrapment into vesicles
(Hao et al. 2002). Therefore, in this study Span 40 and
Span 60 were chosen as the surfactants of choice for the
formation of the non-ionic vesicles.
By inspection of data in Table II it can be concluded
that the entrapment efficiency percentage for niosomes
prepared using Span 60 were significantly higher
(p 5 0.001) than those prepared using Span 40 with
the same molar ratios of Span:CH. This can be
explained by the fact that Span 60 has higher phase
transition temperature (Yoshioka et al. 1994). It was
reported that the higher the transition temperature of
the surfactant the higher the encapsulation efficiency
(Uchegbu and Vyas 1998). Long chain surfactant
produces high entrapment (Hao et al. 2002) and it has
been demonstrated that entrapment efficiency may be
correlated to the hydrophobicity of the alkyl chain of
the sorbitan esters (Manconi et al. 2002). Hence, Span
60 having longer saturated alkyl chain (C18) compared
to Span 40 (C16) produced niosomes with higher
entrapment efficiency. In addition, the length of the
alkyl chain influences the HLB value of the surfactant
mixture which in turn directly affects the drug
entrapment efficiency. The HLB values for Span 40
 504 M. Nasr et al.
(a)
Figure 1. Negative stain electron micrograph of (a) aceclofenac liposomes composed of PC:CH:SA (7:4:1) molar ratio and
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(b) aceclofenac niosomes composed of Span 60:CH (7:4) molar ratio.
and Span 60 are 6.7 and 5, respectively, as provided by
the manufacturer, the lower the HLB of the surfactant
the higher will be the drug entrapment efficiency
(Guinedi et al. 2005).
By comparing the entrapment efficiency percentages
of the prepared aceclofenac liposomal and niosomal
formulations, it can be concluded that aceclofenac
niosomes prepared from Span 60:CH (7:6) molar ratio
showed higher entrapment efficiency than liposomes
For personal use only.
composed of PC:CH of the same molar ratio. This
could be ascribed to the solubilizing effect of the
surfactant used in the preparation of niosomes on
aceclofenac.
Characterization of aceclofenac liposomes and
niosomes
Photomicroscopic analysis and transmisson electron
microscopy. The photomicrographs of aceclofenac
multilamellar liposomes composed of PC:CH:SA
(7:4:1) molar ratio and multilamellar niosomes of
Span 60:CH (7:4) molar ratio revealed the spherical
shape and the multilayered structure of the prepared
liposomes and niosomes that exist in disperse and in
aggregate collections (results not shown). Spherical
niosomes or liposomes display an elastic memory which
has been argued to be of paramount importance for the
success of the carrier-mediated material transport
across the skin (Nasseri 2005).
The electron micrographs of the previous samples are
shown in Figures 1(a) and (b), respectively. They show
the outline and the core of the well identified multi-
lamellar vesicles displaying the retention of sealed
vesicular structure (Morilla et al. 2002).
Vesicle size analysis. Investigation of Table I reveals
that charged liposomes exhibited mean particle
diameter higher than that of the neutral ones. The
frequency distribution curves of particle size data were
unimodal in shape and demonstrated log-normal
distribution of particle size (Manosroi et al. 2002).
The inclusion of a charge inducer in liposomes
(b)
increased the spacing between adjacent bilayers by
repulsion (Taylor et al. 1990; Vyas et al. 1995), thus
increasing the size of the internal aqueous compartment
resulting in the formation of liposomes larger in size
compared to the neutral ones. Furthermore, the
positively charged lipid electrostatically attracts the
drug anion, which may be expected to push phospho-
lipids headgroups apart, thereby increasing the particle
diameter (Hathout et al. 2007). This increase in particle
size would account for the higher entrapment efficiency
of the charged liposomes compared to the neutral ones
(Vyas et al. 1995).
Data in Table II reveals that niosomes prepared using
Span 60 were slightly larger in size than those prepared
using Span 40 (Manconi et al. 2002; Guinedi et al.
2005). Span 60 has a longer saturated alkyl chain
compared to Span 40, which could increase the size of
the vesicle (Manosroi et al. 2003). Larger vesicles are
formed when the hydrophilic portion of the molecule is
decreased relative to the hydrophobic portion, so this
would account for the higher entrapment efficiencies
obtained with Span 60 multilamellar niosomes
(Uchegbu and Duncan 1997), it may also be
attributed to the fact that the increase in alkyl chain
length as the series is ascended from the C12 to the C18
ester would result in an increase in the value of the
critical packing parameter and hence the minimal
permissible vesicle size would increase (Uchegbu and
Florence 1995).
Regarding polydispersity index, a value of 0 indicates
an entirely monodisperse population and a value of 1
indicates a completely polydisperse population
(Zeisig et al. 1996). The prepared vesicular formulations
were mostly heterogenous, which is usually obtained
with vesicles prepared using the thin film hydration
method not followed by sonication or extrusion of the
vesicles (Pereira-Lachataignerais et al. 2006).
The particle size of the prepared vesicles is well suited
for topical delivery through the skin as particles smaller
than 3 mm were found to be distributed into the stratum
corneum and hair follicles (Friedman et al. 1995).

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For personal use only.
Figure 2. DSC thermograms of aceclofenac, plain and drug loaded positively charged liposomes composed of PC:CH:SA
(7:6:1) molar ratio.
Differential scanning calorimetry (DSC). DSC
thermograms of aceclofenac, plain and drug-loaded
multilamellar liposomes composed of 7:6:1 PC:CH:SA
molar ratio as well as multilamellar niosomes composed
of Span 60:CH (7:6) molar ratio are illustrated in
Figures 2 and 3, respectively. These formulations
represented the highest entrapment efficiency among
the prepared liposomes and niosomes, respectively.
A DSC thermogram of aceclofenac showed an
endothermic peak at 150.4 C, which is the reported
melting point of the drug in the literature. (Budavari
et al. 1996). A DSC thermogram of plain liposomes
containing PC:CH:SA in the molar ratio of (7:6:1)
showed a peak endotherm at 102 C. A DSC
thermogram of aceclofenac-loaded liposomes
composed of PC:CH:SA (7:6:1) molar ratio interest-
ingly showed disappearance of the melting endotherm
of aceclofenac and the shift of the endotherm from
102 C to 92.3 C. This may suggest that aceclofenac
can fit into the phospholipids bilayer producing
significant perturbation in the packing characteristics
of the phospholipid membrane and accordingly was
able to reduce the peak of the main transition (Fresta
and Puglisi 1997; Maghraby et al. 2005).
Plain and drug loaded liposomal formulations
showed broad transitions which are characteristic for
lipid mixtures containing cholesterol, signifying good
interaction of all components forming the bilayers of
liposomes (Blume et al. 1993; Nagarsenker et al. 1999).
Vesicularal aceclofenac systems 505
Cholesterol incorporated into the liposomal membrane
had its hydroxyl group oriented towards the aqueous
layer, with the aliphatic chain aligned parallel to the
acyl chains in the centre of lipid bilayers. The presence
of rigid steroid nuclei alongside the carbons of the
phosphatidylcholine chain may reduce the freedom of
movement. This may result in an enhanced stability of
the liposomal membrane (Manosroi et al. 2002).
A DSC thermogram of plain niosomes composed of
7:6 Span 60:CH molar ratio showed an endothermic
peak at 105.5 C. A DSC thermogram of aceclofenac-
loaded niosomes of the same Span 60:CH molar ratio
showed disappearance of the melting endotherm of
aceclofenac and broadening of the endothermic peak
at 103.4 C.
To recapitulate, the absence of the melting
endotherm of aceclofenac and shifting and/or broad-
ening of the endotherms of the lipid bilayer components
of liposomes or surfactant bilayers of niosomes
suggests significant interaction of aceclofenac with
bilayer components and can account for the enhanced
entrapment of aceclofenac into these formulations
(Nagarsenker and Joshi 1997; Hathout et al. 2007).
In vitro drug release studies
In vitro release of aceclofenac from liposomes. The
percentages of drug released after 8 h (T8h) were 61.9%,
53.5% and 43.4% for multilamellar liposomal
 506 M. Nasr et al.
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For personal use only.
Figure 3. DSC thermograms of aceclofenac, plain and drug loaded niosomes composed of Span 60:CH (7:6) molar ratio.
formulations composed of 7:4, 7:6 and 7:7 PC:CH
molar ratios, as shown in Table I. Therefore, on the
basis of the molar ratio of the lipid content, T8h for
the neutral liposomal preparations can be arranged in
the following decreasing order: 7:4 4 7:6 4 7:7 with
significant differences at p 5 0.001. The results could be
due to the fact that the presence of CH in the bilayers at
a temperature above the transition temperature (Tc)
modulates membrane fluidity by restricting the
movement of the relatively mobile hydrocarbon
chains, hence reducing bilayer permeability
(Nagarsenker and Londhe 2003), and condenses the
packing of the phospholipids in the bilayers (Arica et al.
1995), thus decreasing the efflux of the encapsulated
drug (Zhang and Zhu 1999; Fatouros et al. 2001),
resulting in prolonged drug retention (Taylor
et al. 1990).
Data in Table I also shows the effect of charge
inducers on T8h of aceclofenac from multilamellar
liposomal formulations with the lipid content PC:CH
7:4, 7:6 and 7:7 molar ratios. Positively charged
liposomes showed the lowest T8h, where values were
50.7%, 38.2% and 35.8% for liposomal formulations
composed of 7:4:1, 7:6:1 and 7:7:1 PC:CH:SA molar
ratios, respectively. In contrast, negatively charged
liposomes exhibited the highest rates and extent of
drug release. The percentages of drug released after 8 h
for liposomal formulations composed of PC:CH:DP
(7:4:1, 7:6:1 and 7:7:1) molar ratios, respectively, were:
84.4%, 64.8% and 58.7%, with significant differences
at p 5 0.05. Therefore, the order of release of
aceclofenac from multilamellar liposomes may be
arranged in the following order: negatively charged
liposomes 4 neutral liposomes 4 positively charged
liposomes. This order may be due to the electrostatic
attraction forces that may exist between the anionic
drug and positively charged liposomes and vice versa
for negatively charged ones.
In vitro release of aceclofenac from niosomes. From
data in Table II it is obvious that the increase of CH
molar ratio from 7:4 to 7:6 and 7:7 significantly reduced
the efflux of aceclofenac, which is in accordance with
cholesterol membrane stabilizing ability and space
filling action (Uchegbu and Florence 1995; Namedo
and Jain 1999). Cholesterol is known to increase the
rigidity of niosomes (Vyas and Venkatesan 1999) and
abolish the gel-to-liquid phase transition of niosomal
systems resulting in niosomes that are less leaky
(Rogerson et al. 1987), thus decreasing the drug release
from niosomes (Alsarra et al. 2005). Lipophilic drugs
are known to be intercalated within the bilayer
structure of niosomes (Fang et al. 2001b), thus the
release of aceclofenac is sustained from niosomes.
T8h of aceclofenac was 58.2%, 52.1% and 46.1% for
7:4, 7:6 and 7:7 Span 40:CH molar ratios, respectively,
 Vesicularal aceclofenac systems 507

Table III. Determination of the order of release of aceclofenac from different liposomal and niosomal formulations using the coefficient
of determination (r2).

r2

Liposomal or niosomal formulation composition (molar ratio) Zero order First order Diffusion model

PC:CH (7:4) 0.913 0.962 0.986

PC:CH:SA (7:4:1) 0.956 0.979 0.991
PC:CH:DP (7:4:1) 0.928 0.986 0.989
PC:CH (7:6) 0.972 0.986 0.988
PC:CH:SA (7:6:1) 0.953 0.972 0.988
PC:CH:DP (7:6:1) 0.941 0.981 0.988
PC:CH (7:7) 0.969 0.987 0.991
PC:CH:SA (7:7:1) 0.940 0.960 0.988
PC:CH:DP (7:7:1) 0.885 0.930 0.964
Span 40:CH (7:4) 0.901 0.953 0.970
Journal of Microencapsulation Downloaded from informahealthcare.com by University of Calgary on 03/18/13

Span 40:CH (7:6) 0.946 0.973 0.992

Span 40:CH (7:7) 0.925 0.956 0.984
Span 60:CH (7:4) 0.894 0.934 0.977
Span 60:CH (7:6) 0.899 0.932 0.980
Span 60:CH (7:7) 0.947 0.964 0.991

Note: PC: phosphatidylcholine; CH: cholesterol; SA: stearylamine, DP: dicetylphosphate.

while the T8h for 7:4, 7:6 and 7:7 Span 60:CH molar
ratios were 49.4%, 43.3% and 36.2%, respectively. On
the basis of Span:CH ratio, T8h can be arranged in the
following decreasing order: 7:4 4 7:6 4 7:7.
Niosomal formulations prepared using Span 60
For personal use only.

exhibited a significantly slower rate of drug release

compared to Span 40. This can be explained by the fact
that niosomes exhibit an alkyl chain length-dependent
release. The higher the chain length, the lower the
release rate (Devaraj et al. 2002).
Linear regression analysis of the release data reveals
that the drug is released from liposomes and niosomes
by a diffusion controlled mechanism, as shown in
Table III. These results are in agreement with many
research workers who found that the drugs were
released from vesicles by the same mechanism
(Arica et al. 1995; Glavas-Dodov et al. 2002; Hathout
et al. 2007).
Physical stability studies
Positively charged aceclofenac liposomal formulations
composed of PC:CH:SA of 7:4:1 and 7:6:1 molar ratios
and niosomal formulations composed of Span 60:CH of
7:4 and 7:6 molar ratios were considered in this study
as they represented formulations of the highest entrap-
ment efficiency. Physical stability studies on selected
aceclofenac liposomal and niosomal formulations
stored at refrigeration temperature (2–8 C) for a
period of 3 months were conducted by monitoring (a)
the leakage of encapsulated aceclofenac from liposomes
and niosomes and (b) the change in particle size
distribution of the vesicles after storage. In previous
studies pellets of the vesicles were selected for this study
as they showed low leakage rates when stored in the
refrigerator under iso-osmotic conditions (Ozer and
Talsama 1989).
Figure 4. Effect of storage on the percentage aceclofenac
retained in the prepared liposomal and niosomal vesicles.
Effect of storage on drug leakage from
vesicular systems
Drug leakage from the vesicles was evaluated after 15,
30, 45, 60 and 90 days and the results are illustrated in
Figure 4 in terms of percentage of aceclofenac retained
in the vesicular formulations (Patel and Misra 1999).
The percentages of aceclofenac retained in the liposo-
mal formulations after 90 days were 51% and 63% for
liposomal formulations composed of PC:CH:SA (7:4:1)
and PC:CH:SA (7:6:1) molar ratios, respectively. The
leakage of the drug from liposomes may be attributed
to many reasons such as: the fluidity and mobility of the
phospholipid bilayer of the liposomes, the tightness of
interaction of the entrapped materials to the phospho-
lipid bilayers and the size of entrapped molecules
related to their penetrability to the bilayer membrane
(Law et al. 1994).
Moreover, The percentage of aceclofenac retained in
the case of liposomes composed of PC:CH:SA (7:6:1)
was significantly more than those composed of
PC:CH:SA (7:4:1) at p 5 0.001; this may be attributed
 508 M. Nasr et al.
Table IV. Effect of 3 months storage on particle size distribution of selected liposomal and niosomal formulations.
Vesicle type Vesicle composition (molar ratio)
Liposomes PC:CH:SA (7:4:1)
PC:CH:SA (7:6:1)
Niosomes Span 60:CH (7:4)
Span 60:CH (7:6)
to the fact that the presence of cholesterol in the bilayer
of the egg phosphatidylcholine liposomes dramatically
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reduced the leakage of encapsulated material
(Crommelin et al. 1994) by decreasing the rotational
freedom of the phospholipid hydrocarbon chains
(Betageri 1993). This confers structural stability and
gives a more ordered structure bilayer (Patel and
Misra 1999).
On the other hand, the percentages of aceclofenac
retained after a period of 3 months in multilamellar
niosomal formulations composed of Span 60:CH of 7:4
and 7:6 molar ratio were 63.4% and 72.4%,
respectively. Aceclofenac was significantly more
retained in Span 60 niosomes of 7:6 than 7:4 molar
For personal use only.
ratio (p 5 0.05), due to the membrane stabilizing ability
of cholesterol, thus reducing the leakage of drug. The
results indicate that the percentages of aceclofenac
retained in niosomal formulations were higher than
liposomal counterparts.
Effect of storage on particle size distribution of
vesicular formulations
The effect of storage on particle size of liposomal and
niosomal formulations is demonstrated in Table IV.
The results showed (a) slight increase in the vesicle
diameters of the stored liposomal formulations and
(b) slight change in the frequency distribution curves
which was found to be bimodal. This may be due to
slight fusion and aggregation of the liposomes after
storage (Padamwar and Pokharkar 2006). Charge on
lipids is an important parameter influencing liposomal
behaviour. In this way, high surface potential might
increase liposome physical stability by reducing the rate
of aggregation and fusion due to electrostatic repulsion
(Du Plessis et al. 1996; Padamwar and Pokharkar
2006). Niosomal formulations also showed a slight
change in the size and distribution curves on storage
(Gianasi et al. 1997) but this was found to be minimal
compared to liposomes indicating a much more
stable system.
Comparing the results of the physical stability study
of liposomes to that of niosomes, it could be concluded
that niosomes gave better stability. This is possibly
because the problems of phospholipid hydrolysis and
fatty acid peroxidation in liposomes were not observed
for non-ionic surfactants (Fang et al. 2001a).
Distribution modal size (mm)
Fresh vesicles Stored vesicles
1.33 (0.66, 1.6)
1.11 (0.83, 1.29)
0.85 0.86
0.89 1.08
The degradation of non-ionic surfactant is limited so
the replacement of lecithin by non-ionic surfactant has
been considered to overcome this problem. Also the
entrapment of hydrophobic drugs in niosomes is known
to increase their stability (Sihorkar and Vyas 2000).
Previous work has also demonstrated that Span 60
niosomal formulations are generally very stable
(Gianasi et al. 1997) and that drug leaching from
niosomal vesicles made from Span is low due to its high
phase transition temperature and its low permeability
(Vora et al. 1998).
The previous results confirm that niosomes exhibit
superior stability as compared to liposomes (Vora et al.
1998) manifested by better entrapment efficiency and
slight changes in particle size.
In vivo anti-inflammatory study
The liposomal formulations composed of PC:CH:SA of
7:4:1 and 7:6:1 molar ratio and niosomal formulations
composed of Span 60:CH of 7:4 and 7:6 molar ratio
were selected as they represented the formulations
with the highest drug entrapment. Positively charged
liposomes were chosen for the in vivo study for its well
known adhesion to the negatively charged skin, as
reported by Manosroi et al. (2004).
From the results demonstrated in Table V, it can
be concluded that there was overall non-significant
differences in the oedema rate percentage results
between group I (the normal saline control) and
group II (the plain liposomes) or group III (the plain
niosomes) at p 5 0.05, suggesting that the plain
liposomes or niosomes do not possess in vivo anti-
inflammatory effect (Naresh et al. 1994). Differences in
oedema rate percentages between plain liposomes or
niosomes and the saline control were determined using
an unpaired t-test.
Oedema rate of the left foot pad reached its peak at
4 h. The oedema rate and inhibition rate percentages
obtained with different tested preparations are shown in
Table V. All the tested formulations showed better anti-
inflammatory activity than saline control.
Aceclofenac liposomal and niosomal formulations
showed significant (p 5 0.05) percentage inhibition of
oedema compared to control at all time intervals,
suggesting the sustained anti-inflammatory activity of
aceclofenac vesicles throughout the whole time of the
 Journal of Microencapsulation Downloaded from informahealthcare.com by University of Calgary on 03/18/13
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Table V. Oedema rate percentages of plain and aceclofenac-loaded liposomes and niosomes.

Odema rate (%)  SD [Inhibition rate (%)]

Tested formulation 1h 2h 3h 4h 5h 6h 7h 8h

Control saline (Group I) 50.1  8.5 53.3  3.2 63.5  5.7 66.1  3.8 59.3  6.4 52.3  1.9 48.7  3.8 46.4  3.6
Plain liposomes (Group II) 43.2  1.7 51.1  2 60.3  2.7 63.5  0.6 61.6  4.2 59.5  5 48.8  6 40.2  4.9
Plain niosomes (Group III) 50  0.5 54.5  3.4 64.7  1.8 69.7  1.9 56.8  5.4 55.3  1.2 51.1  6.7 39.2  3.5
PC:CH:SA (7:4:1) liposomes (Group IV) 35.7  4.7 38.3  4.5 45  3.6 49.6  0.6 45.8  3.5 44.1  3.9 35.2  3.1 33.1  3.9
[28.7] [28.1] [29.2] [25] [22.8] [15.7] [27.8] [28.6]
PC:CH:SA (7:6:1) liposomes (Group V) 36.1  5.1 37.4  0.5 48.7  5.4 56.2  2.6 52.1  2.6 49  1.6 40.2  0.9 38.7  1.5
[28] [29.9] [23.3] [15] [12.3] [6.3] [17.4] [16.7]
Span 60:CH (7:4) niosomes (Group VI) 34.1  4.7 34.2  1.6 49.7  1.4 50.8  3.4 38.7  3.3 37.4  1.8 31.3  0.8 27.2  3.1
[32] [35.9] [21.7] [23.1] [34.7] [28.5] [35.7] [41.4]
Span 60:CH (7:6) niosomes (Group VII) 34.9  1.9 35  2.9 46.5  2.8 49.1  0.8 44.7  4.8 44.1  2 41.7  2.3 33.1  0.4
[30.5] [34.3] [26.8] [25.7] [24.7] [15.7] [14.3] [28.6]
Marketed product (Group VIII) 26.8  1.9 31.5  2.9 38  1.4 51.1  1.1 40.2  2.3 40.4  1.6 41.3  2.9 42  5.2
[46.5] [40.9] [40.1] [22.7] [32.2] [22.7] [15.2] [9.6]

Statistically significant from saline control Dunnett’s t-test; p 5 0.01, p 5 0.05.
PC: phosphatidylcholine, CH: cholesterol, SA: stearylamine.
Vesicularal aceclofenac systems
509
 510 M. Nasr et al.
study (8 h). Liposomes and niosomes prepared using
PC:CH and Span:CH at a molar ratio of 7:4 showed
better anti-inflammatory activity than those prepared
with the 7:6 molar ratio. This may be attributed to the
fact that, by increasing the CH content, a condensing
effect on the vesicle membranes was exerted, making
them more rigid with a decreased rate of drug
permeation across the skin.
By comparing vesicular formulations to the marketed
product, it was obvious that liposomal and niosomal
tested formulations produced significantly higher anti-
inflammatory activity (lower oedema rate percentage)
after 6 h and 3 h, respectively. The effect was sustained
Journal of Microencapsulation Downloaded from informahealthcare.com by University of Calgary on 03/18/13
until the end of the experiment (8 h), contrary to the
marketed product. It was reported that the vesicle
components together with the drug are deposited into
the skin, thus creating a microreservoir from which the
drug is slowly released (El Ridy and Khalil 1999;
Shahiwala and Misra 2002).
It was obvious that niosomes showed superior anti-
inflammatory activity than liposomes starting from the
fifth hour. This is in accordance with other studies
which revealed that surfactant treated formulations
were superior to phospholipid treated formulations in
facilitating the permeation of drugs, especially those
For personal use only.
prepared using Span 60 (Fang et al. 2001b). Niosomes
loaded with drugs for dermal application are aimed to
preferentially show interactions with the inflamed
(epidermal) tissue without exerting an immediate or
strong systemic action (Junginger et al. 1991; Shahiwala
and Misra 2002).
Conclusion
The liposomal and niosomal formulations proved to
be promising topical systems for the delivery of
aceclofenac as they provided a consistent and
prolonged anti-inflammatory effect and are expected
to minimize the side effects due to the selective build up
of drug concentrations at the site of action. However,
niosomes have proven to be more stable and were
of more efficacious anti-inflammatory effect compared
to liposomes.
Declaration of interest: The authors report no conflicts
of interest. The authors alone are responsible for the
content and writing of the paper.
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